Ethanol is hepatotoxic because it is metabolically converted to compounds that react with proteins to alter their biological activities. Ethanol oxidation by alcohol dehydrogenase (ADH) and cytochrome P-450 2E1 (CYP2E1) catalyze the conversion of ethanol to acetaldehyde. Catalysis by CYP2E1 also leads to enhanced formation of other reactive species including superoxide and hydroxyethyl radicals, which participate in secondary reactions producing hydroxy radicals, peroxynitrite, and lipid peroxides. These metabolites disrupt the activity of proteins, including the proteasome, a multicatalytic enzyme that is indispensable for degrading damaged and misfolded proteins and for tightly controlling the intracellular content of regulatory proteins. Our research has examined the effects of ethanol on proteasome function and its role in hepatic injury, cell regulation and signal transduction. Theses studies have used recombinant Hep G2 (VL-17A) cells that constitutively express both ADH and CYP2E1 and metabolize ethanol comparably to liver cells. When VL-17A cells are exposed to ethanol for extended periods, proteasome activity decreases and this decrease in enzyme activity is dependent on ethanol metabolism. The importance of this finding is that a decline in proteasome activity can result in a variety of effects, among which are a reduction in cell signaling, stabilization of transcription factors and a significant disruption of protein quality control. For our continued investigations, we propose to examine the possible role of the proteasome in ethanol-elicited steatosis, both in cultured cells and in vivo. Furthermore, we present evidence for the ethanol-induced accumulation of protein aggregates in our cultured hepatoma cell model. We hypothesize that ethanol-induced oxidative stress suppresses proteasome activity in liver cells, thereby affecting the levels of Egr-1 and other regulatory proteins involved in steatosis. Additionally, proteasome suppression disrupts protein quality control, thereby causing intracellular protein aggregation in cells. To test this hypothesis, we propose the following Specific Aims: Aim 1 will characterize the ethanol- elicited induction of the lipogenic transcription factors, Egr-1 and SREBP-1 in ethanol metabolizing cultured cells and determine the essentiality of Egr-1 in ethanol-elicited steatosis. Aim 2 will characterize the ethanol- elicited induction of lipogenic transcription factors in mouse liver. Aim 3 will examine whether protein aggregates that form in response to ethanol exposure are effective substrates or inhibitors of the proteasome and whether such aggregates can be eliminated by proteasome activation. PUBLIC HEALTH RELEVANCE: The insights gained from the proposed investigation will expand our knowledge of the mechanisms of ethanol hepatotoxicity. The proposed experimental approach examines a highly regulated cellular proteolytic system that is a potential therapeutic target to alleviate organ damage caused by heavy alcohol consumption.